Anticancer miliusane lactams

ABSTRACT

The present invention is in the field of pharmaceuticals and chemical industries. In particular, the present invention relates to new anticancer miliusane lactam compounds. The present invention also includes its preparation and application method for treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S.patent application Ser. No. 14/927,485, filed on Oct. 30, 2015, which inturn is a continuation-in-part of U.S. patent application Ser. No.13/831,997, filed on Mar. 15, 2013, now U.S. Pat. No. 9,211,333, whichclaims priority of U.S. provisional patent application No. 61/655,990,filed Jun. 5, 2012, all of which are incorporated herein by reference intheir entirety.

FIELD OF INVENTION

The present invention is in the field of pharmaceuticals and chemicalindustries. In particular, the present invention relates to anticanceragents based on miliusane compounds. The present invention also includesits preparation and application method for treating cancer.

BACKGROUND OF INVENTION

Cancer, in one form or another, is a leading cause of death, and claimedthe lives of more than 8.8 million people worldwide in 2015, accordingto the compiled statistics by WHO (WHO:http://www.who.int/mediacentre/factsheets/fs297/en/; retrieved on Apr.15, 2017). It is estimated that annual cancer cases will reach 22million by 2036. Although numerous cancer chemotherapeutics areavailable today, they often have very narrow therapeutic indices andsevere side effects. In addition, cancers can and often do developresistance to many of these drugs. The fact that there currently are nodrugs available that are capable of curing cancer diseases, makes thediscovery and development of new anticancer drugs very much needed andthe undertaking of such studies is imperative.

Miliusanes are a group of compounds containing an oxo-spirodecanesubstructure. A few patents have been published related to the compoundscontaining an oxospirodecane or azaspirodecane or thiaspirocecanesubstructure (US 2009/0318548A1; US2011/021624A1 and WO2011098433A1).

In US 2009/0318548A1, the compounds of formula (VI) are synthesized.However, no anticancer data is reported therein.

In US2011/021624A1, compounds of formula (VII) are synthesized. However,the representative compound is reported to show weak cytotoxicityagainst MDA-MB-435, HCT116, A549 and Hela cancer cells (IC₅₀ values inthe range of 17-42 μM).

In WO2011098433A1, compounds of formula (VIII) are synthesized. Thecompounds have a chemical structure containing a biphenyl group, and arereported to have tumor inhibition activity by inhibiting fatty acidsynthesis.

Thus, miliusanes compounds with more potent anticancer activity and lowtoxicity are needed.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

The present invention relates to anticancer compounds, which aresynthesized based on miliusane. More particularly, the compounds of thepresent invention are derivatives of the core structures of miliusanes.It is a goal of the present invention to provide miliusane derivativeshaving biological activity against cancer, particularly colon cancer,breast cancer, prostate cancer, lung cancer, melanoma, leukemia, braincancer, renal cancer, ovarian cancer, and oral epidermoid cancer.

The present invention provides a series of novel anticancer compoundsbelonging to a cluster of molecules, referred herein to as “miliusanes”,which were isolated from the leaves, twigs and flowers of Miliusasinensis Finet and Gagnep. (Annonaceae) (Zhang H J, Ma C Y, Hung N V,Cuong N M, Tan G T, Santarsiero B D, Mesecar A D, Soejarto D D, PezzutoJ M, Fong H H S. Miliusanes, a class of cytotoxic agents from Miliusasinensis. Journal of Medicinal Chemistry 2006; 49: 693-708).Representative anticancer miliusane compounds are shown below:

The three miliusanes (miliusol, miliusate as well as miliusane I) areevaluated in the NCI 60 cell line panel. When using the NCI automatedCOMPARE analysis, it is observed that the three compounds displayeddifferent GI₅₀ response patterns as compared with other compounds in theNCI database, indicating a unique anticancer mechanism of themiliusanes, which warranted the use of these miliusane compounds forcancer treatment.

U.S. patent application Ser. No. 13/931,997 and U.S. Ser. No. 14/927,485disclose dozens of miliusane derivatives includingN-methyl-2-pyrrolecarboxyl-miliusol, p-dimethylamino-benzoyl-miliusol,4β-(N-phenyl)miliusate, 4α-(N-phenyl)miliusate,4β-(N-benzoyl-N-phenyl)miliusate, 4α-(N-benzoyl-N-phenyl)miliusate,hexahydro-miliusate, 3,4-dihydro-miliusate,2′,3′,6′,7′-tetrahydro-miliusate, 2-hydroxy-3,4-dihydro-miliusate,2-acetoxy-3,4-dihydro-miliusate, 8′-oxo-miliusate, 8′-hydroxy-miliusate,10′-hydroxy-8′-oxo-miliusate, 8′,10′-dihydroxy-miliusate,5β-(p-trimethylammonio-benzoyl)miliusol iodide, and5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide having thefollowing structural formula:

A first aspect of the present invention is a compound based on the corestructures of miliusane for use in the treatment, prevention or delay ofprogression of a cancer in a patient.

A second aspect of the present invention is a pharmaceuticallyacceptable salt or prodrug based on the core structures of miliusane,for use in the treatment, prevention or delay of progression of a cancerin a patient.

A third aspect of the present invention is a pharmaceutical formulationcomprising a compound based on the core structures of miliusane, or apharmaceutically acceptable salt or prodrug thereof, for use in thetreatment, prevention or delay of progression of a cancer in a patient.

A fourth aspect of the present invention is a compound represented bythe formula (IX) or formula (X):

or a pharmaceutically acceptable salt, tautomer, and/or prodrug thereof,wherein

R¹ is alkyl; and

R² is aryl.

In a first embodiment of the fourth aspect of the present inventionthere is provided a compound represented by the formula (IX) or formula(X) wherein said compound is an optically pure stereoisomer.

In a second embodiment of the fourth aspect of the present inventionthere is provided a compound represented by the formula (IX) or formula(X) wherein said compound has formula (IX) or formula (X).

In a third embodiment of the fourth aspect of the present inventionthere is provided a compound represented by the formula (IX) wherein R¹is C₁₋₆ alkyl.

In a fourth embodiment of the fourth aspect of the present inventionthere is provided is a compound represented by the formula (IX) orformula (X) wherein the compounds of formulae (IX) and (X) have therelative stereochemistry depicted below:

In a fifth embodiment of the fourth aspect of the present inventionthere is provided a compound represented by the formula (IX) or formula(X) wherein the compound is selected from N-(n-butyl)-miliusol lactam orN-phenyl-miliusol lactam with the following formula:

or a pharmaceutically acceptable salt and/or prodrug thereof.

In a sixth embodiment of the fourth aspect of the present inventionthere is provided a compound represented by the formula (X) wherein R²is phenyl.

A fifth aspect of the present invention is a pharmaceutical compositioncomprising a compound of the formula (IX) or formula (X) and at leastone pharmaceutically acceptable carrier.

A sixth aspect of the present invention is a method of treating,preventing or delaying the progression of cancer in a subject comprisingadministering a therapeutically effective amount of a compound of theformula (IX) or formula (X) to a subject in need thereof.

A first embodiment of the sixth aspect of the present invention there isprovided a method of treating, preventing or delaying the progression ofcancer in a subject wherein said cancer comprising colon cancer, breastcancer, prostate cancer, lung cancer, oral epidermoid cancer or melanomacancer.

A second embodiment of the sixth aspect of the present invention thereis provided a method of treating, preventing or delaying the progressionof cancer in a subject wherein said subject is a human.

A third embodiment of the sixth aspect of the present invention there isprovided a method of treating, preventing or delaying the progression ofcancer in a subject further comprising the step of administering atherapeutically effective amount of a second anti-cancer agent, whereinsaid compound of claim 1 and said second anti-cancer agent areadministered sequentially or administered simultaneously.

Compounds of the present invention may exist in different forms, such asfree acids, free bases, enantiomers, racemates, diastereomers, estersand other prodrugs, salts and tautomers, and the disclosure includes allvariant forms of these compounds.

The extent of protection includes counterfeit or fraudulent products,which contain or purport to contain a compound of the present inventionirrespective of whether they do in fact contain such a compound andirrespective of whether any such compound is contained in atherapeutically effective amount.

Included in the scope of protection are packages that include adescription or instructions which indicate that the package contains aspecies or pharmaceutical formulation of the present invention and aproduct, which is or comprises, or purports to be or comprise, such aformulation or species. Such packages may be, but are not necessarily,counterfeit or fraudulent.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the present invention are to be understood to be applicableto any other aspect, embodiment or example described herein unlessincompatible therewith.

Those skilled in the art will appreciate that the present inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described.

The present invention includes all such variation and modifications. Thepresent invention also includes all of the steps and features referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.Patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.Patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the present invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the present invention and apply throughout.Unless otherwise defined, all other technical terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which the present invention belongs.

Other aspects and advantages of the present invention will be apparentto those skilled in the art from a review of the ensuing description.

DETAILED DESCRIPTION OF INVENTION

The present invention is not to be limited in scope by any of thespecific embodiments described herein. The following embodiments arepresented for exemplification only.

Definitions

Miliusane and Core Structures

The term “miliusane” as used herein includes reference to a compoundcomprising the basic structure shown as below:

The carbon numbering of miliusane molecule as used herein includesreference to a compound comprising the numbering system shown as below:

The term “the core structure of miliusane” as used herein includesreference to a compound comprising the basic structure shown as below:

In one class of the core structures of miliusane compounds, the methylgroup and the ethyl group form a tetrahydrofuran ring(1-oxa-spiro[4.5]decane and 2-oxa-spiro[4.5]decane).

In the second class of the core structures of miliusane compounds, themethyl group and the ethyl group form a tetrahydro-thiophene ring(1-thia-spiro[4.5]decane and 2-thia-spiro[4.5]decane).

In the third class of the cores structure of miliusane compounds, themethyl group and the ethyl group form a pyrrolidine ring(1-aza-spiro[4.5]decane, 2-aza-spiro[4.5]decane,1-aza-spiro[4.5]dec-1-ene, 2-aza-spiro[4.5]dec-1-ene and2-aza-spiro[4.5]dec-2-ene).

Hydrocarbyl

The term “hydrocarbyl” as used herein includes reference to a moietyconsisting of hydrogen and carbon atoms; such a moiety may comprise analiphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbonatoms. Examples of hydrocarbyl groups include C₁₋₆ alkyl (e.g. C₁, C₂,C₃ or C₄ alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl or tert-butyl); C₁₋₆ alkyl substituted by aryl (e.g. benzyl)or by cycloalkyl (e.g. cyclopropylmethyl); cycloalkyl (e.g. cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl); aryl (e.g. phenyl, naphthyl orfluorenyl) and the like.

Alkyl

The terms “alkyl” and “C₁₋₆ alkyl” as used herein include reference to astraight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbonatoms. This term includes reference to groups such as methyl, ethyl,propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl ortert-butyl), pentyl, hexyl and the like. In particular, the alkyl moietymay have 1, 2, 3 or 4 carbon atoms.

Alkenyl

The terms “alkenyl” and “C₂₋₆ alkenyl” as used herein include referenceto a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6carbon atoms and having, in addition, at least one double bond, ofeither E or Z stereochemistry where applicable. This term includesreference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl,3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, and the like.

Alkynyl

The terms “alkynyl” and “C₂₋₆ alkynyl” as used herein include referenceto a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6carbon atoms and having, in addition, at least one triple bond. Thisterm includes reference to groups such as ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl,3-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.

Alkoxy

The terms “alkoxy” and “C₁₋₆ alkoxy” as used herein include reference to—O-alkyl, wherein alkyl is straight or branched chain and comprises 1,2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1,2, 3 or 4 carbon atoms. This term includes reference to groups such asmethoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy,hexoxy and the like.

Cycloalkyl

The term “cycloalkyl” as used herein includes reference to an alicyclicmoiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be abridged or polycyclic ring system. More often cycloalkyl groups aremonocyclic. This term includes reference to groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, bicyclo[2.2.2]octyl, andthe like.

Aryl

The term “aryl” as used herein includes reference to an aromatic ringsystem comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbonatoms. Aryl is often phenyl but may be a polycyclic ring system, havingtwo or more rings, at least one of which is aromatic. This term includesreference to groups such as phenyl, naphthyl, fluorenyl, azulenyl,indenyl, anthryl, and the like.

Cyclic Group

“Cyclic group” means a ring or ring system, which may be unsaturated orpartially unsaturated but is usually saturated, typically containing 3to 13 ring-forming atoms, for example a 3-, 4-, 5- or 6-membered ring.The ring system may be a bridged or polycyclic ring system. The ring orring system may be substituted with one or more hydrocarbyl groups.Cyclic groups includes carbocyclyl and heterocyclyl moieties.

Carbocyclyl

The term “carbocyclyl” as used herein includes reference to a saturated(e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms. Inparticular, carbocyclyl includes a 3- to 10-membered ring or ring systemand, in particular, 5- or 6-membered rings, which may be saturated orunsaturated. The ring or ring system may be substituted with one or morehydrocarbyl groups. A carbocyclic moiety is, for example, selected fromcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl,bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl,anthryl, and the like.

Heterocyclyl

The term “heterocyclyl” as used herein includes reference to anon-aromatic (e.g. heterocycloalkyl) or an aromatic (e.g. heteroaryl)heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or 16 ring atoms, at least one of which is selected fromnitrogen, oxygen, phosphorus, silicon and sulfur. In particular,heterocyclyl includes a 3- to 10-membered ring or ring system and moreparticularly a 5- or 6-membered ring, which may be saturated orunsaturated. The ring or ring system may be substituted with one or morehydrocarbyl groups.

A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl,1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl,thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl,2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl,imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl,pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl,isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl,pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholino,indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl,indazolyl, triazolyl, tetrazolyl, purinyl, 4/V-quinolizinyl,isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl,benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl,quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl,carbazoiyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl,chromenyl, isochromanyl, chromanyl, and the like.

Heterocycloalkyl

The term “heterocycloalkyl” as used herein includes reference to aheterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2,3, 4, or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorusand sulfur. Heterocycloalkyl can have one or more carbon-carbon doublebonds or carbon-heteroatoms double bonds as long as the ring is notaromatic. The group may be a polycyclic ring system but more often ismonocyclic. This term includes reference to groups such as azetidinyl,pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl,imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl,thiomorpholinyl, quinolizidinyl, and the like. The ring or ring systemmay be substituted with one or more hydrocarbyl groups.

Heteroaryl

The term “heteroaryl” as used herein includes reference to an aromaticheterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or16 ring atoms, at least one of which is selected from nitrogen, oxygenand sulfur. The group may be a polycyclic ring system, having two ormore rings, at least one of which is aromatic, but is more oftenmonocyclic. The ring or ring system may be substituted with one or morehydrocarbyl groups. This term includes reference to groups such aspyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl,imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl,purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl,phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl,indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl, and thelike.

Halogen

The term “halogen” as used herein includes reference to F, Cl, Br, or I.

Halogen Containing Moiety

The expression “halogen containing moiety” as used herein includesreference to a moiety comprising 1 to 30 plural valence atoms selectedfrom carbon, nitrogen, oxygen and sulfur which moiety includes at leastone halogen. The moiety may be hydrocarbyl for example C₁₋₆ alkyl orC₁₋₆ alkoxy, or carbocyclyl for example aryl.

Substituted

The term “substituted” as used herein in reference to a moiety meansthat one or more, especially up to 5, more especially 1, 2 or 3, of thehydrogen atoms in said moiety are replaced independently of each otherby the corresponding number of the described substituents. The term“optionally substituted” as used herein means substituted orun-substituted. It will, of course, be understood that substituents areonly at positions where they are chemically possible, the person skilledin the art being able to decide (either experimentally or theoretically)without inappropriate effort whether a particular substitution ispossible.

Enantiomer

The term “enantiomer” as used herein means one of two stereoisomers thathave mirror images of one another.

Racemate

The term “racemate” as used herein means a mixture of equal amounts ofenantiomers of a chiral molecule.

Diastereomer

The term “diastereomer” as used herein means one of a class ofstereoisomers that are not enantiomers, but that have differentconfigurations at one or more of the equivalent chiral centers. Exampleof diasteromers are epimers that differ in configuration of only onechiral center.

Stereoisomer

The term “stereoisomer” as used herein means one of a class of isomericmolecules that have the same molecular formula and sequence of bondedatoms, but different three-dimensional orientations of their atoms inspace.

Tautomers

The term “tautomer” means isomeric molecules that readily interconvertby a chemical reaction. The reaction commonly results in the migrationof a hydrogen atom, which results in a switch of a single bond andadjacent double bond.

Prodrug

A prodrug is a medication that is administered as an inactive (or lessthan fully active) chemical derivative that is subsequently converted toan active pharmacological agent in the body, often through normalmetabolic processes.

Independently

Where two or more moieties are described as being “each independently”selected from a list of atoms or groups, this means that the moietiesmay be the same or different. The identity of each moiety is thereforeindependent of the identities of the one or more other moieties.

Embodiments of the present invention are described below. Preferredfeatures of each aspect of the present invention are as for each of theother aspects mutatis mutandis. Moreover, it will be appreciated thatthe features specified in each embodiment may be combined with otherspecified features, to provide further embodiments.

Compounds of the Present Invention

In an exemplary embodiment, the present invention provides compounds offormula (I), or (II):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵and R¹⁶ are each independently selected from hydrogen, halogen or amoiety comprising 1 to 30 plural valence atoms selected from carbon,nitrogen, oxygen and sulfur; or these groups may be taken together withthe carbon atoms to which they are attached to form one or more cyclicgroups which is optionally substituted with halogen or a moietycomprising 1 to 30 plural valence atoms selected from carbon, nitrogen,oxygen and sulfur; R¹ and R², R³ and R⁴, R⁵ and R⁶, R⁷ and R⁸, R⁹ andR¹⁰, R¹¹ and R¹², R¹³ and R¹⁴ or R¹⁵ and R¹⁶ may be taken together withthe carbon atoms to which they are attached to form one or more carboxylgroups (C═O); or while one of R¹ and R², R³ and R⁴, R⁵ and R⁶, R⁷ andR⁸, R⁹ and R¹⁰, R¹¹ and R¹², R¹³ and R¹⁴ or R¹⁵ and R¹⁶ is hydrogen,halogen, hydrocarbyl or alkoxy, the other one of R¹ and R², R³ and R⁴,R⁵ and R⁶, R⁷ and R⁸, R⁹ and R¹⁰, R¹¹ and R¹², R¹³ and R¹⁴ or R¹⁵ andR¹⁶ is selected from R¹⁷, —OR¹⁷, —C(O)R¹⁷ and —C(O)OR¹⁷; R¹⁷ isindependently selected from hydrogen, halogen, trifluoromethyl, cyano,nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R¹⁸,heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R¹⁸,—(CH₂)_(k)-heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R¹⁸,—OR¹⁹, —C(O)R²⁰, —C(O)N(R¹⁹)R²⁰, —C(O)OR¹⁹, —OC(O)R¹⁹, —S(O)₂R¹⁹,—S(O)₂N(R¹⁹)R²⁰, —N(R¹⁹)R²⁰, —N(R¹⁹)N(R¹⁹)R²⁰, —N(R¹⁹)C(O)R²⁰ and—N(R¹⁹)S(O)₂R²⁰; wherein k is an integer between 1 and 6 (e.g. 1, 2 or3); R¹⁸ is independently selected from halogen, trifluoromethyl, cyano,nitro, oxo, ═NR¹⁹, —OR¹⁹, —C(O)R²⁰, —C(O)N(R¹⁹)R²⁰, —C(O)OR¹⁹,—OC(O)R²⁰, —S(O)₂R¹⁹, —S(O)₂N(R¹⁹)R²⁰, —N(R¹⁹)R²⁰, —N(R¹⁹)N(R¹⁹)R²⁰,—N(R¹⁹)C(O)R²⁰ and —N(R¹⁹)S(O)₂R²⁰; R¹⁹ and R²⁰ are each independentlyhydrogen or selected from hydrocarbyl, heterocyclyl or—(CH₂)_(k)-heterocyclyl, which is optionally substituted with 1, 2, 3, 4or 5 substituents independently selected from halogen, cyano, amino,hydroxy, C₁₋₆ alkyl and C₁₋₆ alkoxy; wherein k is an integer between 1and 6 (e.g. 1, 2 or 3); X is oxygen or sulfur; R is hydrogen or selectedfrom hydrocarbyl, heterocyclyl or —(CH₂)_(k)-heterocyclyl, which isoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from halogen, cyano, amino, hydroxy, C₁₋₆ alkyl and C₁₋₆alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3); dashedline “

” denotes a single or double bond; or a pharmaceutically acceptable saltor prodrug thereof.

In certain embodiments, the compounds of the present invention arerepresented by formula (IX) or formula (X):

or a pharmaceutically acceptable salt, tautomer, and/or prodrug thereof,wherein R¹ is alkyl; and R² is aryl.

In certain embodiments the compound is an optically pure stereoisomer.

In certain embodiments, the compound has formula (X).

In certain embodiments, the compound of formula (X) has the relativestereochemistry indicated below:

In certain embodiments, the compound of formula (X) has the absolutestereochemistry indicated below:

In certain embodiments, R² is phenyl.

In certain embodiments, the compound has formula (IX).

In certain embodiments, the compound of formula (IX) has the relativestereochemistry indicated below:

In certain embodiments, the compound of formula (IX) has the absolutestereochemistry indicated below:

In certain embodiments, R¹ is C₁₋₆ alkyl, C₂₋₆ alkyl, C₃₋₆ alkyl, orC₃₋₅ alkyl. In certain embodiments, R¹ is C₄ alkyl. In certainembodiments, R¹ is n-butyl.

In certain embodiments, the compound is a tautomer of the compound ofFormula (IX) or (X).

In certain embodiments, the compound has the same relativestereochemistry at the C-1, C-1′, and C-5 carbons as miliusane.

In certain embodiments, the compound has the same absolutestereochemistry at the C-1, C-1′, and C-5 carbons as miliusane.

In certain embodiments, the compound is an enantiomer.

In certain embodiments, the compound is a racemate.

In certain embodiments, the compound is a diastereomer.

In certain embodiments, the compound is a tautomer.

The present invention also provides a pharmaceutical compositioncomprising a compound of the present invention and at least onepharmaceutical acceptable carrier.

It should be noted that compounds having one of the following formulaeshould not be the compound of the present invention and should beremoved from the compound of the present invention:

wherein Ac is acetyl group (CH₃C═O), and Me is methyl group (CH₃);Compounds having the following formula (V) are excluded in the presentinvention:

wherein R¹═R²═R³═R⁴═R⁵═H; or R¹═R²═R³═R⁴═H, R⁵═OCH₃; or R¹═R³═R⁴═R⁵═H,R²═OCH₃; or R¹═R²═R⁴═R⁵═H, R³═OCH₃; or R¹═R²═R⁴═H, R³═R⁵═OCH₃; orR¹═R⁵═OCH₃, R²═R³═R⁴═H; or R¹═R⁴═R⁵═H, R²═R³═OCH₃; or R¹═R³═R⁵═H,R²═R⁴═OCH₃; or R¹═R⁵═H, R²═R³═R⁴═OCH₃; or R¹═R⁴═R⁵═H, R² and R³ takentogether with the carbon atoms to which they are attached to form acyclic [1,3]dioxole group; or R¹═R²═R³═R⁴═H, R⁵═CH₃; or R¹═R³═R⁴═R⁵═H,R²═CH₃; or R¹═R²═R⁴═R⁵═H, R³═CH₃; or R¹═R²═R³═R⁴═H, R⁵═F; orR¹═R³═R⁴═R⁵═H, R²═F; or R¹═R²═R⁴═R⁵═H, R³═F; or R¹═R²═R³═H, R⁴═R⁵═F; orR¹═R²═R⁴═H, R³═R⁵═F; or R¹═R³═R⁴═H, R²═R⁵═F; or R¹═R⁵═F, R²═R³═R⁴═H; orR¹═R⁴═R⁵═H, R²═R³═F; or R¹═R³═R⁵═H, R²═R⁴═F; or R¹═R²═R³═R⁴═H, R⁵═Cl; orR¹═R³═R⁴═R⁵═H, R²═Cl; or R¹═R²═R⁴═R⁵═H, R₃═Cl; or R¹═R²═R⁴═H, R³═R⁵═Cl;or R¹═R⁵═Cl, R²═R³═R⁴═H; or R¹═R⁴═R⁵═H, R²═R³═Cl; or R¹═R³═R⁵═H,R²═R⁴═Cl; or R¹═R²═R³═R⁴═H, R⁵═Br; or R¹═R³═R⁴═R⁵═H, R²═Br; orR¹═R²═R⁴═R⁵═H, R³═Br; or R¹═R²═R³═R⁴═H, R⁵═I; or R¹═R²═R⁴═R⁵═H, R³═I;Compounds having the following formula (VI) are excluded in the presentinvention:

wherein X¹ and X² are carbon atoms either joined by double bond orjoined by a single bond constituents of an epoxide ring or ahydroxyethylene moiety; X³ and X⁴ are carbon atoms either joined bydouble bond or joined by a single bond constituents of an epoxide ringor a hydroxyethylene moiety; R¹ is selected from the group consisting ofbranched alkyl chains, unbranched alkyl chains, cycloalkyl groups,aromatic groups, alcohols, ethers, amines, and substituted orunsubstituted ureas, esters, aldehydes and carboxylic acids; R² isselected from the group consisting of H, OH and NHR³, wherein R³ is anitrogen protecting group;Compounds having the following formula (VII) are excluded in the presentinvention:

wherein R¹, R³ and R⁴ are selected from the group consisting of branchedalkyl chains, unbranched alkyl chains, cycloalkyl groups, aromaticgroups, alcohols, ethers, amines, and substituted or unsubstitutedureas, esters, aldehydes and carboxylic acids; m is an integer between 0and 5;Compounds having the following formula (VIII) are excluded in thepresent invention:

wherein Z is oxygen or sulfur or nitrogen substituted with D group; Drepresents hydrogen or represents a C₁-C₆-alkyl orC₁-C₆-alkoxy-C₁-C₆-alkyl radical or represents a C₃-C₇-cycloalkyl or 4-to 7-membered monocyclic heterocyclyl radical, where the radicalsmentioned may optionally be mono- or polysubstituted by identical ordifferent substituents selected from the group consisting of halogen andhydroxyl and C₁-C₃-alkyl, halo-C₁-C₃-alkyl, or C₁-C₃-alkoxy; Xrepresents halogen, nitro or cyano or represents an optionallymonohalogen- or polyhalogen-substituted C₁-C₆-alkyl, C₁-C₆-alkoxy,C₁-C₆-alkoxy-C₁-C₆-alkoxy, C₃-C₇-cycloalkyl or aC₃-C₇-cycloalkyl-C₁-C₆-alkoxy radical, and W and Y independently of oneanother represent hydrogen, nitro, cyano or halogen or represent anoptionally monohalogen- or polyhalogen-substituted C₁-C₆-alkyl,C₁-C₆-alkoxy or C₃-C₇-cycloalkyl radical, and V¹, V² and V³independently of one another represent hydrogen, halogen, nitro or cyanoor represent a C₁-C₆-alkyl, halo-C₁-C₆-alkyl, C₁-C₆-alkoxy,halo-C₁-C₆-alkoxy, C₁-C₆-alkylthio, C₁-C₆-alkylsulphinyl,C₁-C₆-alkylsulphonyl, C₁-C₆-alkoxy-C₁-C₆-alkyl, C₃-C₁₀-cycloalkylradical or represent a monocyclic heterocycloalkyl radical, and/orV¹ and V² together with the carbon atoms to which they are attached forma saturated or unsaturated cycle T¹ which optionally contains at leastone further heteroatom and has 4 to 7-ring atoms and whose ring-formingatoms may be mono- or polysubstituted by identical or differentsubstituents selected from the group consisting of halogen and aC₁-C₆-alkyl radical.

Examples of the compounds of the present invention include those shownbelow. It will of course be appreciated that, where appropriate, eachcompound may be in the form of the free compound, an enantiomer, an acidor base addition salt, or a prodrug.

The present invention provides 17 compounds with potent anti-canceractivity and synthesis thereof. The compounds of the present inventionare synthesized and evaluated for their anticancer activity, namely4β-(N-phenyl)miliusate, 4α-(N-phenyl)miliusate,4β-(N-benzoyl-N-phenyl)miliusate, 4α-(N-benzoyl-N-phenyl)miliusate,hexahydro-miliusate, 3,4-dihydro-miliusate,2′,3′,6′,7′-tetrahydro-miliusate, 2-hydroxy-3,4-dihydro-miliusate,2-acetoxy-3,4-dihydro-miliusate, 8′-oxo-miliusate, 8′-hydroxy-miliusate,10′-hydroxy-8′-oxo-miliusate, 8′,10′-dihydroxy-miliusate,5β-(p-trimethylammonio-benzoyl)miliusol iodide,5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide,N-(n-butyl)-miliusol lactam and N-phenyl-miliusol lactam. The chemicalformulae of the compounds of the present invention are as follows:

Examples

Synthesis of Novel Miliusane Derivatives.

The two natural miliusane compounds (miliusol and miliusate), isolatedfrom Miliusa balansae Finet & Gagnep. (Annonaceae), were used as thestarting compounds to prepare new miliusane derivatives. By usingdifferent synthetic methods, 17 new miliusane analogs are synthesized.

These new miliusane compounds have been evaluated for their anticanceractivity against a panel of cancer cell lines comprising KB, HCT116,LNCaP, A549, MCF-7 and A375. 4β-(N-Phenyl)miliusate demonstrates cellkilling activity with IC₅₀ values ranging from 0.2-2.0 μM.4α-(N-Phenyl)miliusate demonstrated cell killing activity with IC₅₀values ranging from 1.1-6.5 μM. 4β-(N-Benzoyl-N-phenyl)miliusatedemonstrates cell killing activity with IC₅₀ values ranging from 0.5-2.5μM. 4α-(N-Benzoyl-N-phenyl)miliusate demonstrates cell killing activitywith IC₅₀ values ranging from 5.0-9.8 μM.2′,3′,6′,7′-Tetradehydro-miliusate demonstrates cell killing activitywith IC₅₀ values ranging from 1.2-6.8 μM. 8′-Oxo-miliusate demonstratescell killing activity with IC₅₀ values ranging from 3.6-5.5 μM.8′-Hydroxy-miliusate demonstrates cell killing activity with IC₅₀ valuesranging from 4.2-8.9 μM. 10′-Hydroxy-8′-oxo-miliusate demonstrates cellkilling activity with IC₅₀ values ranging from 2.5-4.9 μM.5β-(p-Trimethylammonio-benzoyl)miliusol iodide demonstrates cell killingactivity with IC₅₀ values ranging from 0.05-1.2 μM.5β-(p-Dimethyl-allyl-ammonio-benzoyl)miliusol bromide demonstrates cellkilling activity with IC₅₀ values ranging from 0.1-1.5 μM.N-(n-butyl)-miliusol lactam demonstrates cell killing activity with IC₅₀values ranging from 0.5-4.2 μM. 5β-(p-Trimethylammonio-benzoyl)miliusoliodide, 5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide, and4β-(N-phenyl)miliusate demonstrate more potent cell killing activitythan that of miliusol. All other compounds show no cell killing activityat a concentration of 20 μM.

Since 4β-(N-phenyl)miliusate shows better cell killing activity thanthat of miliusol in the in vitro evaluation system, the compound isfurther evaluated for its in vivo antitumor activity in HCT116 xenograftmouse model study. In the experiment, HCT116 tumors in mice treated with4β-(N-phenyl)miliusate grow much slower than those mice treated withvehicle. After 21 days administration, the measured average tumor size(L×W×W) is suppressed by 30.5% with treatment of 4β-(N-phenyl)miliusateat the dose of 20 mg/kg (p<0.01) in comparison with the vehicle controlgroup (paclitaxel, the clinically used anticancer drug, shows 23.3%inhibition of tumor growth at the dose of 10 mg/kg). In addition, noweight loss is observed for the mice in the 4β-(N-phenyl)miliusatetreatment group. On the contrary, 3 out of 10 mice died for thepaclitaxel group. These results clearly indicate the superior antitumorefficacy of 4β-(N-phenyl)miliusate with low toxicity in comparison withthe clinically used drug paclitaxel.

General Method for Preparation of 4-Amino Substituted MiliusaneDerivatives.

Bismuth nitrate [Bi(NO₃)₃] (70 mg, 0.5 eq) is added to a mixture of anamine such as aniline (1.35 g, 50 eq) and a miliusane such as miliusate(100 mg, 0.29 mmol) in dichloromethane (CH₂Cl₂, 2 mL). The reactionmixture is stirred at room temperature for 48 hours. CH₂Cl₂ (30 mL) isadded to the reaction mixture, and the CH₂Cl₂ solution is filteredthrough filter paper to remove Bi(NO₃)₃. The filtrate is washed withsaturated NaHCO₃ (2×10 mL) and brine (1×10 mL), and dried over Na₂SO₄.The CH₂Cl₂ solution is concentrated, and is further purified usingchromatography on a silica gel column eluting with a mixture of solventsuch as petroleum ether:EtOAc (ethyl acetate) 4:1 to afford 4-aminosubstituted miliusane derivative(s). Unreacted, partially reacted orunwanted compounds as disclosed herein can be removed from the reactionproduct.

The new molecules 4β-(N-phenyl)miliusate and 4α-(N-phenyl)miliusate arethus synthesized (see Scheme 1).

General Method for Preparation of Amino Substituted Miliusane AmideDerivatives.

To a solution of an amino substituted miliusane derivative such as4α-(N-benzyl)miliusate and 4β-(N-benzyl)miliusate (30 mg, 0.068 mmol) inanhydrous pyridine (5 mL) is added benzoyl chloride (BzCl) (19 mg, 2 eq)and a catalytic amount of 4-dimethylaminopyridine (DMAP). The reactionis stirred overnight at room temperature, and quenched with H₂O (30 mL).The reaction mixture is then extracted with ethyl acetate (3×20 mL). Thecombined ethyl acetate solution is washed with H₂O (20 mL) and brine (20mL), and dried over Na₂SO₄. The solution is then concentrated in vacuo,and chromatographed on a silica gel column, eluted with a solvent systemsuch as petroleum ether:EtOAc 2:1 to afford amino substituted miliusaneamide derivative. Unreacted, partially reacted or unwanted compounds asdisclosed herein can be removed from the reaction product.

The new molecules 4β-(N-benzoyl-N-phenyl)miliusate, and4α-(N-benzoyl-N-phenyl)miliusate are thus synthesized (see Schemes 2 and3).

General Method for Preparation of Hexahydro-Miliusane Derivatives.

A solution of a miliusane such as miliusate (20 mg, 0.058 mmol) in EtOAc(5 mL) is treated with 10% of the catalyst palladium on activatecharcoal (Pd/C) (5 mg). The resulting black suspension is stirred underhydrogen (H₂) (1 atmosphere) at room temperature for 3 hours. Thecatalyst is removed by filtration through Celite and washed with EtOAc(20 mL). The filtrate is evaporated in vacuo and the residue issubjected to chromatography separation on silica gel, eluted with asolvent system such as petroleum ether:EtOAc 4:1 to affordhexahydro-miliusane derivative. Unreacted, partially reacted or unwantedcompounds as disclosed herein can be removed from the reaction product.

The new molecule hexahydro-miliusate is thus synthesized (see Scheme 4).

General Method for Preparation of 3,4-Dihydro-Miliusane Derivatives.

A solution of a miliusane such as miliusate (10 mg, 0.029 mmol) in EtOAc(3 mL) is treated with 10% Pd/C (5 mg). The resulting black suspensionis stirred under H₂ (1 atmosphere) at −78° C. for 48 hours. The catalystis removed by filtration through Celite and washed with EtOAc (20 mL).The filtrate is evaporated in vacuo and the residue is subjected tochromatography separation on silica gel, eluted with a solvent systemsuch as petroleum ether:EtOAc 4:1 to afford the 3,4-dihydro-miliusanederivative. Unreacted, partially reacted or unwanted compounds asdisclosed herein can be removed from the reaction product.

The new molecule 3,4-dihydro-miliusate is thus synthesized (see Scheme5).

General Method for Preparation of 2′,3′,6′,7′-Tetrahydro-MiliusaneDerivatives.

To a solution of a miliusane such as hexahydro-miliusate (20 mg, 0.056mmol) in toluene:DMSO (2:1, 0.1 M) is added 2-iodoxybenzoic acid (IBX)(20.7 mg, 1.3 eq). The resulting solution is heated to 100° C. forstirring 3 hours. The reaction mixture is then diluted with CH₂Cl₂ (30mL), and washed sequentially with 5% NaHCO₃ (1×10 mL), H₂O (1×10 mL),and brine (1×10 mL), followed by removal of solvent in vacuo, leading tocrude products, which can be purified using silica gel columnchromatography to afford the 2′,3′,6′,7′-tetrahydro-miliusanederivative. Unreacted, partially reacted or unwanted compounds asdisclosed herein can be removed from the reaction product.

The new molecules 2′,3′,6′,7′-tetrahydro-miliusate is thus synthesized(see Scheme 6).

General Method for Preparation of 2-Hydroxy-Miliusane Derivatives.

To a stirred solution of a miliusane such as miliusate (20 mg, 0.058mmol) in MeOH (5 mL) is added sodium borohydride (NaBH₄) (2.2 mg, 1 eq)at 0° C. The reaction mixture is stirred at 0° C. for 1 hour, quenchedwith saturated ammonium chloride (NH₄Cl) (10 mL), and then extractedwith EtOAc (3×20 mL). The combined EtOAc solution is washed sequentiallywith H₂O (10 mL) and brine (10 mL), dried over Na₂SO₄, and concentratedin vacuo. The reaction mixture is subjected to chromatography separationon a silica gel column, eluted with a solvent system such as petroleumether:EtOAc 2:1 to afford the 2-hydroxy-miliusane derivative. Unreacted,partially reacted or unwanted compounds as disclosed herein can beremoved from the reaction product.

The new molecule 2-hydroxy-3,4-dihydro-miliusate is thus synthesized(see Scheme 7).

General Method for Preparation of Ester Derivatives of2-Hydroxy-Miliusanes.

A solution of a 2-hydroxy-miliusane such as 2-hydroxy-3,4-dihydro-miliusate (8 mg, 0.023 mmol) in CH₂Cl₂ (5 mL) is treated withtriethylamine (TEA) (16.0 μL, 5 eq), anhydrous acetic acid (Ac₂O) (6.5μL, 3 eq) and a catalytic amount of DMAP. The reaction mixture isstirred at room temperature for 3 hours, quenched with saturated NaHCO₃(20 mL), and then extracted with EtOAc (3×10 mL). The combined EtOAcsolution is washed with H₂O (10 mL) and brine (10 mL), dried overNa₂SO₄, and concentrated in vacuo. The mixture is chromatographed on asilica gel column, eluting with petroleum ether:EtOAc 4:1 to afford theester derivative of 2-hydroxy-miliusane. Unreacted, partially reacted orunwanted compounds as disclosed herein can be removed from the reactionproduct.

The new molecule 2-acetoxy-3,4-dihydro-miliusate is thus synthesized(see Scheme 8).

General Method for Preparation of Aldehyde and Hydroxy Derivatives onthe Side Chain of Miliusanes.

To a 25-mL flask is introduced 16 mg (0.5 eq) of selenium dioxide(SeO₂), 10 mL of CH₂Cl₂ and 80 μL (2 eq) of 70% tert-butyl hydroperoxide(TBHP). After the mixture is stirred for 0.5 hours at 25° C. (waterbath), a miliusane such as miliusate (100 mg, 0.29 mmol) is added inseveral minutes. The mixture is stirred at 25° C. for 48 hours. Thesolvent is then removed in vacuo, and the residue is subjected to silicagel column chromatography separation to give side chain aldehyde andhydroxy derivatives of miliusanes. Unreacted, partially reacted orunwanted compounds as disclosed herein can be removed from the reactionproduct.

The new molecules 8′-oxo-miliusate, 8′-hydroxy-miliusate,10′-hydroxy-8′-oxo-miliusate, and 8′,10′-dihydroxy-miliusate are thussynthesized (see Scheme 9).

General Method for Preparation of Miliusol Ester Derivatives.

The solution of 5.0 mg of miliusane such as miliusol (0.016 mmol) in 1.0mL of dry pyridine is pipetted into a solution of selected acyl chloridereagent such as p-dimethyl-benzyol chloride (0.2 mmol) in 1.0 mL of drypyridine at 0° C. The reaction is allowed to proceed at 0° C. for 2hours, and additional 20 hours at room temperature. The reaction productis evaporated in vacuo to dryness to afford a mixture, which issubjected to a silica gel column chromatography to afford theacyl-miliusane ester. Unreacted, partially reacted or unwanted compoundsas disclosed herein can be removed from the reaction product. Themolecule 5β-(p-dimethylamino-benzoyl)miliusol is thus synthesized (seeScheme 10).

General Method for Preparation of Miliusane Ammonium Salts.

A chloroform (CHCl₃) solution of an amino group containing miliusanederivative such as 5β-(p-dimethylamino-benzoyl)miliusol (10 mg, 0.022mmol) and an alkyl halide such as methyl iodide or allyl bromide (10 mL)is refluxed under N₂ atmosphere for 72 hours. Products can be detectedby TLC, HPLC and LC-MS. The reaction product is evaporated in vacuo todryness to afford a mixture, which is subjected to a Si gel columnseparation to afford miliusane ammonium salt product. Unreacted,partially reacted or unwanted compounds as disclosed herein can beremoved from the reaction product.

The new molecules 5β-(p-trimethylammonio-benzoyl)miliusol iodide and5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide are thussynthesized (see Schemes 11 and 12).

General Method for Preparation of Miliusane Lactam.

A mixture of a miliusane such as miliusol (6.1 mg, 0.02 mmol), a primaryamine such as n-butylamine (5.9 μL, 0.06 mmol) or aniline (5.5 μL, 0.06mmol) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF₄, 3.7μL, 0.02 mmol) is vortexed in a sealed vessel for 30 seconds prior tomicrowave irradiation at 220° C. for 35 minutes. The cooled reactionmixture is then diluted with 5 mL of EtOAc and washed with saturatedaqueous NH₄Cl. The collected EtOAc solution is concentrated in vacuo,and purified over a silica gel column to afford miliusane lactamproduct. Unreacted, partially reacted or unwanted compounds as disclosedherein can be removed from the reaction product.

The new molecules N-(n-butyl)-miliusol lactam and N-phenyl-miliusollactam are thus synthesized (see Schemes 13 and 14).

4β-(N-Phenyl)miliusate is obtained as a white powder with a molecularformula of C₂₆H₃₃NO₅ determined by positive HRESIMS and NMR studies. ¹HNMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal ofCDCl₃ at δ 7.24 ppm) phenyl protons [7.18 (2H, brt, J=7.5), 6.79 (1H,brt, J=6.7), 6.60 (2H, brs)], 5.58 (1H, d, J=10.6, H-1′), 5.48 (1H, brs,H-5), 4.96 (1H, brd, J=10.8, H-2′), 4.94 (1H, m, H-6′), 3.81 (1H, brdt,J=13.1, 3.5, H-4), 3.57 (1H, d, J=18.1, H-7β), 2.76 (1H, brdd, J=13.1,3.1, H-3α), 2.66 (1H, dd, J=15.7, 2.7, H-6β), 2.48 (1H, brt, J=13.1,H-3β), 2.24 [3H, s, OC(═O)CH₃], 2.00 (1H, d, J=18.0, H-7α), 2.02 (4H, m,H₂-4′ and H₂-5′), 1.84 (3H, d, J=1.3 Hz, CH₃-9′), 1.75 (1H, dd, J=15.6,3.3, H-6α), 1.63 (3H, s, CH₃-8′), 1.55 (3H, s, CH₃-10′). HRTOF positiveESIMS m/z calcd for C₂₆H₃₃NO₅: 440.2432 [M+1]⁺. found: 440.2425 [M+1]⁺.

4α-(N-Phenyl)miliusate is obtained as a white powder with a molecularformula of C₂₆H₃₃NO₅ determined by positive HRESIMS and NMR studies. ¹HNMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal ofCDCl₃ at δ 7.24 ppm) phenyl protons [7.21 (2H, brt, J=7.8), 6.79 (3H,m)], 5.81 (1H, d, J=10.6, H-1′), 5.38 (1H, brq, J=2.9, H-5), 5.01 (1H,brd, J=10.7, H-2′), 4.95 (1H, m, H-6′), 4.04 (1H, m, H-4), 3.57 (1H, d,J=18.1, H-7β), 2.81 (1H, dd, J=14.0, 4.6, H-3β), 2.40 (1H, brd, J=15.3,H-6β), 2.39 (1H, brd, J=13.2, H-3α), 2.23 [3H, s, OC(═O)CH₃], 2.16 (1H,dd, J=15.4, 3.8, H-6α), 2.08 (1H, d, J=18.3, H-7α), 2.04 (4H, m, H₂-4′and H₂-5′), 1.86 (3H, d, J=1.3 Hz, CH₃-9′), 1.66 (3H, s, CH₃-8′), 1.57(3H, s, CH₃-10′). HRTOF positive ESIMS m/z calcd for C₂₆H₃₃NO₅: 440.2432[M+1]⁺. found: 440.2436 [M+1]⁺.

4β-(N-Benzoyl-N-phenyl)miliusate is obtained as a white powder with amolecular formula of C₃₃H₃₇NO₆ determined by positive HRESIMS and NMRstudies ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to thesignal of CDCl₃ at δ 7.24 ppm) aromatic protons [7.06-7.25 (8H, m),6.93-7.00 (2H, m)], 5.66 (1H, brtd, J=8.3, 4.9, H-5), 5.22 (1H, brd,J=10.0, H-2′), 5.10 (1H, d, J=10.0, H-1′), 4.99 (1H, m, H-6′), 4.60 (1H,brtd, J=8.0, 5.9, H-4), 3.33 (1H, d, J=17.4, H-7β), 2.87 (1H, dd,J=16.6, 5.8, H-3α), 2.74 (1H, dd, J=16.6, 8.4, H-3β), 2.42 (1H, d,J=17.5, H-7α), 2.19 (1H, dd, J=13.9, 4.9, H-6β), 2.11 (1H, dd, J=14.0,8.9, H-6α), 2.09 [3H, s, OC(═O)CH₃], 2.05 (4H, m, H₂-4′ and H₂-5′), 1.76(3H, d, J=1.3 Hz, CH₃-9′), 1.64 (3H, s, CH₃-8′), 1.57 (3H, s, CH₃-10′).HRTOF positive ESIMS m/z calcd for C₃₃H₃₇NO₆: 544.2694 [M+1]⁺. found:544.2690 [M+1]⁺.

4α-(N-Benzoyl-N-phenyl)miliusate is obtained as a white powder with amolecular formula of C₃₃H₃₇NO₆ determined by positive HRESIMS and NMRstudies. ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference tothe signal of CDCl₃ at δ 7.24 ppm) aromatic protons [7.06-7.22 (8H, m),6.97-7.01 (2H, m)], 5.94 (1H, brq, J=2.2, H-5), 5.58 (1H, d, J=10.6,H-1′), 5.00 (1H, brddd, J=14.8, 3.9, 2.7, H-4), 4.92 (1H, brdd, J=10.6,1.0, H-2′), 4.88 (1H, m, H-6′), 3.57 (1H, d, J=18.0, H-7β), 2.79 (1H,dd, J=14.9, 13.1, H-3β), 2.55 (1H, brdd, J=15.6, 2.9, H-6β), 2.48 (1H,brdd, J=13.0, 3.9, 1.2, H-3α), 2.05 [3H, s, OC(═O)CH₃], 1.96 (4H, m,H₂-4′ and H₂-5′), 1.88 (1H, dd, J=15.6, 3.4, H-6α), 1.98 (1H, d, J=18.2,H-7α), 1.67 (3H, d, J=1.3 Hz, CH₃-9′), 1.62 (3H, s, CH₃-8′), 1.52 (3H,s, CH₃-10′). HRTOF positive ESIMS m/z calcd for C₃₃H₃₇NO₆: 544.2694[M+1]⁺. found: 544.2699 [M+1]⁺.

Hexadehydro-miliusate is obtained as a white powder with a molecularformula of C₂₀H₃₂O₅ determined by positive HRESIMS and NMR studies. ¹HNMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal ofCDCl₃ at δ 7.24 ppm) 5.29 (1H, brquintet, J=3.2, H-5), 5.01 (1H, brdd,J=11.6, 2.1, H-1′), 3.43 (1H, d, J=17.9, H-7β), 2.41-2.64 (3H, m), 2.28(1H, m), 2.13 [3H, s, OC(═O)CH₃], 2.04 (1H, m), 2.01 (1H, d, J=17.9,H-7α), 1.83 (1H, dd, J=15.2, 3.7, H-6α), 1.68 (1H, m), 1.27-1.56 (4H,m), 0.94-1.28 (5H, m), 0.88 (3H, d, J=6.7 Hz), 0.84 (3H, d, J=6.6 Hz),0.83 (3H, d, J=6.6 Hz). HRTOF positive ESIMS m/z calcd for C₂₄H₃₂O₅:353.2323 [M+1]⁺. found: 353.2318 [M+1]⁺.

3,4-Didehydro-miliusate is obtained as a white powder with a molecularformula of C₂₀H₂₈O₅ determined by positive HRESIMS and NMR studies. ¹HNMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal ofCDCl₃ at δ 7.24 ppm) 5.74 (1H, d, J=10.6, H-1′), 5.27 (1H, brquintet,J=3.0, H-5), 4.98 (1H, brdd, J=10.6, 1.0, H-2′), 4.94 (1H, m, H-6′),3.54 (1H, d, J=18.0, H-7β), 2.55 (1H, brdt, J=15.3, 3.0), 2.42 (1H,brtd, J=14.6, 6.1), 2.30 (1H, brdd, J=13.4, 5.8, 2.9), 2.25 (1H, m),2.15 [3H, s, OC(═O)CH₃], 2.01 (4H, m, H₂-4′ and H₂-5′), 2.00 (1H, d,J=18.0, H-7α), 1.96 (2H, m), 1.82 (3H, d, J=1.3 Hz, CH₃-9′), 1.64 (3H,s, CH₃-8′), 1.55 (3H, s, CH₃-10′). HRTOF positive ESIMS m/z calcd forC₂₀H₂₈O₅: 349.2010 [M+1]⁺. found: 349.2005 [M+1]⁺.

2′,3′,6′,7′-Tetradehydro-miliusate is obtained as a white powder with amolecular formula of C₂₆H₃₃NO₅ determined by positive HRESIMS and NMRstudies. ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference tothe signal of CDCl₃ at δ 7.24 ppm) 6.87 (1H, brddd, J=10.2, 4.2, 1.8,H-4), 6.16 (1H, brdd, J=10.2, 1.1, H-3), 5.52 (1H, brqd, J=4.0, 1.1,H-5), 4.72 (1H, brdd, J=10.8, 2.6, H-1′), 3.20 (1H, d, J=17.5, H-7β),2.37 (1H, brdtd, J=14.9, 3.9, 0.9), 2.26 (1H, brdt, J=14.7, 4.2), 2.25(1H, d, J=17.6, H-7α), 2.11 [3H, s, OC(═O)CH₃], 1.83 (1H, dd, J=15.2,3.7, H-6α), 1.40-1.60 (2H, m), 0.93-1.40 (7H, m), 0.88 (3H, d, J=6.7Hz), 0.83 (6H, d, J=6.5 Hz). HRTOF positive ESIMS m/z calcd forC₂₀H₃₀O₅: 351.2166 [M+1]⁺. found: 351.2156 [M+1]⁺.

2-Hydroxy-3,4-didehydro-miliusate is obtained as a white powder with amolecular formula of C₂₀H₃₀O₅ determined by positive HRESIMS and NMRstudies. ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference tothe signal of CDCl₃ at δ 7.24 ppm) 5.24 (1H, brd, J=10.4, H-2′), 4.99(1H, m, H-6′), 4.82 (1H, d, J=10.5, H-1′), 4.76 (1H, brseptet, J=4.7,H-5), 3.70 (1H, brs, H-2), 2.47 (1H, d, J=17.3, H-7β) 2.33 (1H, brdd,J=17.3, 1.4, H-7α), 2.07-2.24 (5H, m), 2.02 [3H, s, OC(═O)CH₃], 1.98(1H, brt, J=11.4), 1.77-1.92 (4H, m), 1.74 (3H, d, J=1.3 Hz, CH₃-9′),1.67 (3H, s, CH₃-8′), 1.60 (3H, s, CH₃-10′). HRTOF positive ESIMS m/zcalcd for C₂₀H₃₀O₅: 351.2166 [M+1]⁺. found: 351.2160 [M+1]⁺.

2-Acetoxy-3,4-didehydro-miliusate is obtained as a white powder with amolecular formula of C₂₂H₃₂O₆ determined by positive HRESIMS. HRTOFpositive ESIMS m/z calcd for C₂₂H₃₂O₆: 393.2272 [M+1]⁺. found: 393.2266[M+1]⁺.

8′-Oxo-miliusate is obtained as a white powder with a molecular formulaof C₂₀H₂₄O₆ determined by positive HRESIMS and NMR studies. ¹H NMR (400MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal of CDCl₃ atδ 7.24 ppm) 9.37 (1H, s, H-8′), 6.81 (1H, brddd, J=10.2, 4.0, 0.8, H-4),6.33 (1H, brtq, J=7.1, 1.3, H-6′), 6.00 (1H, brdd, J=10.2, 1.2, H-3),5.55 (1H, brq, J=4.2, H-5), 5.45 (1H, d, J=9.9, H-1′), 5.19 (1H, brdq,J=9.9, 1.3, H-2′), 3.31 (1H, d, J=17.6, H-7β), 2.43 (1H, brt, J=7.5,H-5′a), 2.41 (1H, brt, J=7.3, H-5′b), 2.36 (1H, brddd, J=14.6, 4.3, 0.9,H-6β), 2.27 (1H, d, J=17.6, H-7α), 2.24 (1H, dd, J=14.7, 5.5, H-6α),2.19 (2H, brt, J=7.3, H₂-4′), 2.13 [3H, s, OC(═O)CH₃], 1.70 (3H, brd,J=1.1 Hz, CH₃-10′), 1.69 (3H, d, J=1.3 Hz, CH₃-9′). HRTOF positive ESIMSm/z calcd for C₂₀H₂₄O₆: 361.1646 [M+1]⁺. found: 361.1640 [M+1]⁺.

8′-Hydroxy-miliusate is obtained as a white powder with a molecularformula of C₂₀H₂₆O₆ determined by positive HRESIMS and NMR studies. ¹HNMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference to the signal ofCDCl₃ at δ 7.24 ppm) 6.83 (1H, brddd, J=10.3, 4.3, 1.3, H-4), 6.08 (1H,brdd, J=10.2, 1.2, H-3), 5.57 (1H, d, J=10.1, H-1′), 5.56 (1H, m, H-5),5.17 (1H, m, H-6′), 5.05 (1H, brdq, J=10.1, 1.0, H-2′), 4.01 (1H, ABbrd,J=13.5, H-8′a), 3.95 (1H, ABbrd, J=13.4, H-8′b), 3.43 (1H, d, J=17.7,H-7β), 2.44 (1H, brddd, J=15.0, 3.2, 1.4, H-6β), 2.23 (1H, dd, J=15.1,5.5, H-6α), 2.18 (1H, d, J=17.7, H-7α), 2.14 [3H, s, OC(═O)CH₃], 2.12(2H, m, H₂-5′), 2.07 (2H, m, H₂-4′), 1.65 (3H, d, J=1.3 Hz, CH₃-9′),1.61 (3H, s, CH₃-10′). HRTOF positive ESIMS 172/Z calcd for C₂₀H₂₆O₆:363.1802 [M+1]⁺. found: 363.1808 [M+1]⁺.

10′-Hydroxy-8′-oxo-miliusate is obtained as a white powder with amolecular formula of C₂₀H₂₄O₇ determined by positive HRESIMS and NMRstudies. ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference tothe signal of CDCl₃ at δ 7.24 ppm) 9.42 (1H, s, H-8′), 6.82 (1H, brddd,J=10.2, 3.9, 0.8, H-4), 6.45 (1H, brt, J=7.4, H-6′), 6.02 (1H, brdd,J=10.2, 1.2, H-3), 5.56 (1H, brq, J=4.2, H-5), 5.45 (1H, d, J=9.9,H-1′), 5.21 (1H, brdq, J=9.9, 1.2, H-2′), 4.35 (1H, ABd, J=12.8,H-10′a), 4.30 (1H, ABd, J=12.7, H-10′b), 3.32 (1H, d, J=17.6, H-7β),2.53 (1H, brtd, J=7.6, 2.3, H-5′a), 2.51 (1H, brt, J=7.4, H-5′b), 2.39(1H, brddd, J=14.6, 4.3, 0.7, H-6β), 2.26 (1H, d, J=17.7, H-7α), 2.23(2H, brt J=7.1, H₂-4′), 2.23 (1H, overlap, H-6α), 2.14 [3H, s,OC(═O)CH₃], 1.70 (3H, d, J=1.3 Hz, CH₃-9′). HRTOF positive ESIMS m/zcalcd for C₂₀H₂₄O₇: 377.1595 [M+1]⁺. found: 377.1600 [M+1]⁺.

8′,10′-Dihydroxy-miliusate is obtained as a white powder with amolecular formula of C₂₀H₂₆O₇ determined by positive HRESIMS and NMRstudies. ¹H NMR (400 MHz, CDCl₃, J in Hz) δ (in ppm with reference tothe signal of CDCl₃ at δ 7.24 ppm) 9.42 (1H, s, H-8′), 6.83 (1H, brddd,J=10.2, 4.1, 1.1, H-4), 6.10 (1H, brdd, J=10.2, 1.1, H-3), 5.56 (1H,brq, J=4.1, H-5), 5.50 (1H, d, J=10.0, H-1′), 5.31 (1H, brt, J=7.1,H-6′), 5.08 (1H, brdq, J=10.0, 1.2, H-2′), 4.25 (1H, ABd, J=12.8,H-10′a), 4.21 (1H, ABd, J=12.8, H-10′b), 4.19 (1H, ABd, J=13.0, H-8′a),4.15 (1H, ABd, J=13.0, H-8′b), 3.36 (1H, d, J=17.7, H-7β), 1.63 (2H,brs, 2×OH), 2.40 (1H, brddd, J=14.9, 3.7, 1.1, H-6β), 2.23 (1H, dd,J=14.9, 5.5, H-6α), 2.22 (1H, d, J=17.9, H-7α), 2.17 (1H, brt, J=6.7,H-5′a), 2.16 (1H, brt, J=7.2, H-5′b), 2.13 [3H, s, OC(═O)CH₃], 2.07 (2H,brt J=6.7, H₂-4′), 1.65 (3H, d, J=1.3 Hz, CH₃-9′). HRTOF positive ESIMSm/z calcd for C₂₀H₂₆O₇: 379.1751 [M+1]⁺. found: 379.1750 [M+1]⁺.

5β-(p-Trimethylammonio-benzoyl)miliusol iodide is obtained as a whitepowder with a molecular formula of C₂₈H₃₆NO₅I determined by positiveHRESIMS. HRTOF positive ESIMS m/z calcd for C₂₈H₃₆NO₅: 466.2588 [M]⁺.found: 466.2579 [M]⁺.

5β-(p-Dimethyl-allyl-ammonio-benzoyl)miliusol bromide is obtained as awhite powder with a molecular formula of C₃₀H₃₈NO₅Br determined bypositive HRESIMS. HRTOF positive ESIMS m/z calcd for C₃₀H₃₈NO₅: 492.2745[M]⁺. found: 492.2735 [M]⁺.

N-(n-Butyl)-miliusol lactam is obtained as a white powder with amolecular formula of C₂₂H₃₃NO₃ determined by positive HRESIMS. HRTOFpositive ESIMS m/z calcd for C₂₂H₃₄NO₃: 360.2539 [M+1]⁺. found: 360.2530[M+1]⁺.

N-Phenyl-miliusol lactam is obtained as a white powder with a molecularformula of C₂₄H₂₉NO₃ determined by positive HRESIMS. HRTOF positiveESIMS m/z calcd for C₂₄H₃₄NO₃: 380.2226 [M+1]⁺. found: 380.2235 [M+1]⁺.

Cell Culture Panel Bioassays.

All pure compounds of the present invention are evaluated against humancancer cell lines using a cytotoxicity screening panel. Cytotoxicityassays involving oral epidermoid (KB), colon (HCT116), prostate (LNCaP),breast (MCF-7), lung (A549) and melanoma (A375) carcinoma cell lines,are performed using sulforhodamine B according to established protocols(Zhang H J, Ma C Y, Hung N V, Cuong N M, Tan G T, Santarsiero B D,Mesecar A D, Soejarto D D, Pezzuto J M, Fong H H S. Miliusanes, a classof cytotoxic agents from Miliusa sinensis. Journal of MedicinalChemistry 2006; 49: 693-708; and Jutiviboonsuk A, Zhang H J, Tan G T, MaC M, Hung N V, Cuong N M, Bunyapraphatsara N, Soejarto D D, Fong H H S.Bioactive constituents from the roots of Bursera tonkinensis.Phytochemistry 2005; 66: 2745-2751.). KB cells are maintained in DMEMmedium. LNCaP cells are maintained in RPMI1640 medium with hormone-free10% heat-activated FBS (fetal bovine serum) supplemented with 0.1 nMtestosterone. MCF-7 cells are maintained and assayed in MEME mediumcontaining 10 mg/L of insulin. HCT116 cells are maintained in McCoy's 5Amedium supplemented with 10% fetal bovine serum. A549 and A375 cells aremaintained in RPMI-1640 medium supplemented with 10% FCS. Serialdilutions of the compounds are prepared using 10% aqueous DMSO assolvent. The 190 μL cell suspension (3×10⁴ cells in 1 ml media) isincubated with 10 μL sample solutions, in triplicate, in 96-well tissueculture plate at 37° C. in a humidified atmosphere of 5% CO₂ in air for72 hours. 10 μL 10% aqueous DMSO is used as control group. Then thecells are fixed to plastic substratum by the addition of 100 μL cold 20%aqueous trichloroacetic acid and washing with water after incubation at4° C. for 30 min. After staining cells with 100 μL of 0.4%sulforhodamine B in 1% aqueous AcOH for 30 min, unbound dye is removedby rinsing with 1% aqueous AcOH. The bound dye is solubilized with 200μL 10 mM unbuffered Tris base, pH 10, and the optical density ismeasured at 515 nm using an ELISA plate reader. The average data areexpressed as a percentage, relative to the control. The IC₅₀ values, thedose that inhibit cell growth by 50%, are calculated using nonlinearregression analysis (percent survival versus concentration).

Antitumor Animal Study.

4β-(N-Phenyl)miliusate shows potent cell killing activity in our invitro evaluation system. In order to determine its antitumor activity,HCT116 xenograft animal study is used to evaluate the anticanceractivity of 4β-(N-phenyl)miliusate in comparison with the clinicallyused anticancer drug paclitaxel. All animal studies are approved andperformed according to Animal Care and Use Guidelines of the AnimalEthics Committee at Hong Kong Baptist University and performed followingAnimal Care and Use guidelines set by NIH (National Institute of Health,USA). BALB/c nude mice, SPF class, male or female, 7-8 weeks old, arepurchased from Charles River Laboratories. Before the experiment, themice are kept for one week of acclimatization to SPF class laboratoryconditions. 4β-(N-Phenyl)miliusate was tested for its antitumor activityagainst HCT116 cancer cells using a number of nude mice (Balc/nu/nu,female) in comparison of paclitaxel. HCT116 cancer cells aresubcutaneously implanted with 5×10⁶ cells in the rear flank of eachmouse. After 10 days, solid tumors with average size of about 80 mm³appeared at the implanted sites. The mice are then divided into threegroups: one dose (20 mg/kg: 10 mice) group of 4β-(N-phenyl)miliusate,one dose (10 mg/kg: 10 mice) of paclitaxel and one dose of vehicle(negative control: 10 mice). Every other day injections at i.p. sitesare scheduled for 21 days. Weights of mice and tumor diameters aremeasured twice a week until the end of the experiment. The tumor size inmm³ is calculated by the formula: tumor size=length×width×height(L×W×H).

Having now fully described the present invention in some detail by wayof illustration and examples for purposes of clarity of understanding,it will be obvious to one of ordinary skill in the art that the same canbe performed by modifying or changing the present invention within awide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the present invention or anyspecific embodiment thereof, and that such modifications or changes areintended to be encompassed within the scope of the appended claims. Theterms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of the presentinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of the present invention.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms.

When a group of materials, compositions, components or compounds isdisclosed herein, it is understood that all individual members of thosegroups and all subgroups thereof are disclosed separately. When aMarkush group or other grouping is used herein, all individual membersof the group and all combinations and subcombinations possible of thegroup are intended to be individually included in the disclosure. Everyformulation or combination of components described or exemplified hereincan be used to practice the present invention, unless otherwise stated.Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure. In the disclosure and the claims, “and/or” meansadditionally or alternatively. Moreover, any use of a term in thesingular also encompasses plural forms.

All references cited herein are hereby incorporated by reference intheir entirety to the extent that there is no inconsistency with thedisclosure of this specification. Some references provided herein areincorporated by reference to provide details concerning sources ofstarting materials, additional starting materials, additional reagents,additional methods of synthesis, additional methods of analysis,additional biological materials, additional cells, and additional usesof the present invention. All headings used herein are for convenienceonly. All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe present invention pertains, and are herein incorporated by referenceto the same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated by reference. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

If desired, the different functions discussed herein may be performed ina different order and/or concurrently with each other. Furthermore, ifdesired, one or more of the above-described functions may be optional ormay be combined.

While the foregoing invention has been described with respect to variousembodiments and examples, it is understood that other embodiments arewithin the scope of the present invention as expressed in the followingclaims and their equivalents. Moreover, the above specific examples areto be construed as merely illustrative, and not limitative of thereminder of the disclosure in any way whatsoever. Without furtherelaboration, it is believed that one skilled in the art can, based onthe description herein, utilize the present invention to its fullestextent. All publications recited herein are hereby incorporated byreference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention discloses new anticancer agents based on themiliusane natural products. The present invention also includes itspreparation and application method for treating cancer.

What is claimed is:
 1. A compound represented by the formula (IX) orformula (X):

or a pharmaceutically acceptable salt, tautomer, and/or prodrug thereof,wherein R¹ is alkyl; and R² is aryl.
 2. The compound of claim 1, whereinsaid compound is an optically pure stereoisomer.
 3. The compound ofclaim 1, wherein said compound has formula (IX).
 4. The compound ofclaim 1, wherein said compound has formula (X).
 5. The compound of claim3, wherein R¹ is C₁₋₆ alkyl.
 6. The compound of claim 4, wherein R² isphenyl.
 7. The compound of claim 1, wherein the compounds of formula(IX) and (X) have the relative stereochemistry depicted below:


8. The compound of claim 1, wherein the compound is selected fromN-(n-butyl)-miliusol lactam or N-phenyl-miliusol lactam with thefollowing formula:

or a pharmaceutically acceptable salt and/or prodrug thereof.
 9. Apharmaceutical composition comprising a compound of claim 1 and at leastone pharmaceutically acceptable carrier.
 10. A method of treating cancerin a subject comprising administering a therapeutically effective amountof a compound of claim 1 to a subject in need thereof, wherein saidcancer is colon cancer, breast cancer, prostate cancer, lung cancer,epidermoid cancer, or melanoma cancer.
 11. The method of claim 10,wherein said subject is a human.
 12. The method of claim 10, furthercomprising the step of administering a therapeutically effective amountof a second anti-cancer agent, wherein said compound of claim 1 and saidsecond anti-cancer agent are administered sequentially or administeredsimultaneously.